Molecular and Hematological Characterization of Hemoglobin H Disease in the Bengali Population of Kolkata,India

Introduction

Hemoglobin H (HbH) disease is the severest form of α-thalassemia compatible with postnatal life and is caused by mutations involving three α-globin genes (Chen et al., 2000; Higgs and Bowden, 2001; Waye et al., 2001; Chui et al., 2003; Boona et al., 2004; Sen and Talukdar, 2005). This disease most frequently results from the interaction of α⁺ and α⁰ thalassemia determinants.

The syndrome of HbH disease is usually that of thalassemia intermedia, although there is considerable variability in the clinical and hematological severity (Skogerboe et al., 1992). The degree of hemolysis is more severe in HbH patients with nondeletional α-globin gene mutations (Liu et al., 1994).

The predominant features are anemia (2.6-13.3 g/dL) with jaundice, hepatosplenomegaly, and the presence of HbH (0.8%-40%) occasionally accompanied by Hb Bart's in the peripheral blood. Blood film shows marked red cell morphological changes, including severe hypochromia, microcytosis, and target cell formation. Patients with the -α/α^Tα genotype tend to be more anemic, with markedly higher levels of HbH and a more severe clinical course than those with the deletional (- -/-α) forms of HbH disease (Viprakasit and Tanphaichitr, 2002).

The earliest case of HbH was recorded in a Bengali subject from Kolkata (Swarup et al., 1963). Many cases of HbH disease were documented worldwide (Chen et al., 2000; Kanavakis et al., 2000; Wenning et al., 2000; Liu et al., 2000; Waye et al., 2001; Higgs, 2001; Zorai et al., 2002, 2003; Eng et al., 2005; Li et al., 2005; Origa et al., 2006; Chan et al., 2007; Sura et al., 2007). Some cases of HbH diseases have been found in the newborn in West Bengal, and in Bombay, 2% and 4% of cord samples contained Hb Bart's (Chouhan et al., 1970; Mitra, 1983). Sen et al. (2004) reported two cases of HbH disease from Bengali families in Kolkata, West Bengal.

α-Thalassemia among the tribes has been reported in many regions of India and neighboring countries (Reddy et al., 1995; Sakai et al., 2000). Incidence of α-thalassemia among the tribes of Northeast India has been reported by our group earlier (Sen et al., 2005).

Screening of α-thalassemia among the nontribe Bengali population in cases of unexplained anemia has not been reported earlier. This is the first report on the incidence of HbH disease with molecular analysis of common mutations among the nontribe Bengali in West Bengal. An attempt has been made to detect HbH disease among the selected cases referred to our department, Thalassemia Counseling Unit, Vivekananda Institute of Medical Sciences, from the hematology outpatient department (OPD). Initially, patients were referred to the Out Patient Department of Hematology, Ramakrisna Mission Seva Pratisthan Hospital, Kolkata, for various hematological disorders. Of these patients, 105 patients with unexplained anemia (the reason for anemia was not known) were referred to the Thalassemia Counseling Unit or further investigation. These patients showed low hematocrit values (mean corpuscular volume [MCV] <78 fL; mean corpuscular hemoglobin [MCH] <28 pg) and most of them were asymptomatic. Alpha thalassemia is asymptomatic, so it is very difficult to diagnose by the clinicians. We did hemoglobin electrophoresis and found HbH disease in 17 of the patients.

Molecular analysis was also carried out in the identified HbH cases to detect the type of common α-thalassemia mutation.

Materials and Methods

A total of 105 subjects of nontribe Bengali population of both sexes were referred to our laboratory, Thalassemia Counseling Unit, Vivekananda Institute of Medical Science of Ramakrishna Mission Seva Pratisthan, for investigation of unexplained anemia. Blood, collected by venesection in ethylenediaminetetraacetic acid vacutainers, was subjected to the following tests: complete hemogram, total count (TC), differential count (DC), MCV, MCH, and mean corpuscular hemoglobin concentration (MCHC), using an automated cell counter Sysmex K-1000.

Stained slides were screened for the detection of Heinz body inclusions (multiple blue-green spherical inclusions) on exposure to brilliant cresyl blue (Dacie and Lewis, 1994).

Agarose gel electrophoresis was used to detect the HbH band. DNA was extracted using a commercial kit (Quiagen blood mini kit) and modified multiplex polymerase chain reaction technique was used for the detection of common -α3.7 and -α4.2 thalassemia deletions at α-globin gene (Shajii et al., 2000).

Result

Various hematological parameters, the results of hemoglobin electrophoresis, and common α-thalassemia mutations (-3.7/-4.2/South East Asian [- -SEA]) in patients with HbH disease are shown in Table 1.

Of 105 cases referred to our department, 17 (16.19%) showed the presence of HbH band in hemoglobin electrophoresis. Screening for the two most common α-globin deletions by multiplex polymerase chain reaction revealed a homozygous rightward pattern (-α3.7/-α3.7) in three persons and homozygous leftward deletion (-α4.2/-α4.2) in one person. Five appeared as compound heterozygote (-α3.7/-α4.2) and one was reported to show the presence of (-3.7α/- -SEA) mutation. The other seven cases did not show the common α mutations. Number 12 was prior identified as a β-thalassemia carrier in hemoglobin electrophoresis along with HbH. The β-thalassemia mutation was identified as IVS 1-5 (G→C) by the amplified refractory mutation system (ARMS) method.

Discussion

To investigate the current prevalence of HbH disease in the Bengali population of West Bengal, we studied 105 cases who had unexplained anemia. The age group of the studied population ranged from 1 to 77 years and 90 of them had low hemoglobin levels (<11 g/dL). Of these patients, HbH disease was documented in 17 persons (16.19%) by hemoglobin electrophoresis. Most of them seemed to be clinically asymptomatic. Screening of the two most common deletion mutations on the α-globin gene (-α3.7 and -α4.2) showed a high prevalence among the diagnosed persons as 10 of 17 cases showed homozygous leftward deletions (-α4.2/-α4.2), homozygous rightward (-α3.7/-α3.7), and double heterozygote states (-α4.2/-α3.7).

One patient of the 17 cases was Chinese in origin. A (- -SEA) mutation study revealed the presence of this mutation in him along with -α3.7 in heterozygous state. Because the (- -SEA) mutation is not common among Bengali, this mutation study had not been carried out in them. Number 12 showed the presence of IVS 1-5 (G→C) β-thalassemia mutation, whereas the common α deletion was absent.

The seven cases that showed the absence of the two deletional mutations should be investigated further for the presence of other deletional/nondeletional mutation. In this study, we screened for mainly two common α-thalassemia deletion mutations. But, a third mutation in HbH disease should be taken into consideration for future study.

As α-thalassemia is asymptomatic in origin, its diagnosis is very difficult. α-Thalassemia carriers with one or two deletions on the α-globin gene can lead a life of a normal individual, but if this is ignored, the future consequences of this genetic disease could be fatal. Social awareness, genetic testing and counseling, and screening among the newborn should be mandatory for the eradication of this disease.